The long-term goal of this proposal is to gain detailed understanding of how the diaphragm - the main muscle of respiration - rapidly weakens in response to mechanical unloading and of the mechanisms whereby the giant elastic protein titin influences this response. The diaphragm is a unique muscle in that it is constantly subjected to mechanical loading. Recent work suggests that diaphragm strength is remarkably sensitive to mechanical unloading, as occurs during mechanical ventilation in the ICU. How unloading affects diaphragm strength is poorly understood. Increasing this understanding is critically important: within hours, diaphragm unloading during mechanical ventilation causes diaphragm weakness in ICU patients, which leads to difficulties in weaning patients from ventilatory support and contributes to mortality. Th search for the molecular triggers for the development of diaphragm weakness during mechanical unloading is ongoing. The potential role of mechano-sensor proteins, that link diaphragm unloading to protein turnover, is unexplored but is an exciting and novel concept that needs to be studied. A candidate mechano-sensor is titin, a giant elastic protein that has been suggested to sense mechanical stress and link this to trophic signalling pathways. The elucidation of titin's role in diaphragm trophicity and in diaphragm weakness during mechanical ventilation is central to this grant proposal. Aim 1 will critically test whether titin affects musle trophicity. I will use unilateral diaphragm denervation (UDD), a condition that is clinically important and that presents itself as a great tool for this work as it induces rapid hypertrophy of the denervated hemidiaphragm due to cyclic passive stretch. I will study UDD in two novel titin KO mouse models: one in which titin stiffness is increased through deletion of Ig domains (Ig KO) and another in which titin stiffness is decreased through deletion of the titin splice factor rbm20 (Rbm20 KO). I anticipate that the hypertrophic response following UDD is exaggerated in Ig KO mice and blunted in Rmb20 KO mice, and that this response is mediated by altered titin signaling. Aim 2 will study whether low titin stiffness protects the diaphragm from weakening during mechanical ventilation-induced unloading and will use a rat model with low titin stiffness. If titin-based mechano-sensing mediates the response of the diaphragm to mechanical unloading, then I anticipate that low titin stiffness, by preconditioning the diaphragm to reduced mechanosensing, blunts this response. Aim 3 will study the mechanistic basis for diaphragm weakness in mechanically ventilated ICU patients using, for the first time, diaphragm fibers isolated from biopsies of mechanically ventilated ICU patients. The goal is to investigate whether the findings of animal studies extrapolate to patients. The innovation of this proposal lies in the novel research foci with innovative guiding hypotheses, its novel mouse models, unique diaphragm biopsies from mechanically ventilated ICU patients, and its novel experimental tools. The proposal's integrative approach is expected to lead to a significant step forward in our understanding of diaphragm function and the role of titin therein.